Article Figures & SI

Figures

Minimum evolution phylogeny for fibril A collagen proteins as obtained with JTT plus Γ distances (α = 0.906). Numbers indicate bootstrap scores for each node, based on 1,000 replicates, whereas branch lengths are proportional to expected replacements per site. This tree is rooted by sea urchin ColP2α. Equally and unequally weighted MP, ML, and BP also place the two lamprey sequences together at the base of the Col2α1 clade, with bootstrap scores or a posterior probability of 93%, 77%, 71%, and 87%, respectively. In contrast to this consistent support for a Col2α1 grouping, this ME tree is unique among the different phylogenies in its placement of the root along the Col5 clade. This discrepancy over the root is illustrated with the BP phylogeny and discussed further in Fig. 6.

Col2α1a and Col2α1b expression during lamprey development. Whole mount in situ hybridization of lamprey embryos at stages 23 (A–D), 25 (E), 26 (G–I), and 30 (F and J–L). Anterior is to the right in (A, C, J, and L); All sections are transverse with dorsal to the top. (A and B) Col2α1a expression is evident in the somites and within the dermatome and sclerotome. Expression in the midline is restricted to floor plate and hypochord (B). (C and D) Col2α1b is expressed in the floor plate, notochord, hypochord, and dorsal endoderm (asterisk in D). (E) Col2α1a expression in the dermatome, sclerotome, floor plate, and hypochord. (F) Col2α1a expression in a prevertebral condensation (arrows). (G–I) Sections through the hindbrain (G), mid-trunk (H), and tail (I) show an anterior to posterior retraction of the Col2α1b domain in the notochord, hypochord, and floor plate. (J) Col2α1a is expressed throughout the branchial skeleton (boxed) and posterior to the oral cavity. (K) Col2α1a expression in a stack of chondrocytes in a branchial bar. (L) Col2α1b is not detected in the branchial skeleton. bb, branchial basket; d, dermatome; fp, floor plate; h, hypochord; n, notochord; oc, oral cavity; s, sclerotome; so, somite.

Extended majority-rule consensus tree for the BP analysis of the chordate SoxE proteins. Numbers indicate posterior probabilities for groups with >50% credibility and for those clades that are combinable with this first set. Branch lengths are proportional to the means of the posterior probability densities for their expected replacements per site. This tree is rooted by sea squirt SoxE. Equally and unequally weighted MP, ME, and ML also place the previously undescribed lamprey sequence at the base of the Sox9 clade, with bootstrap scores of 86%, 74%, 28%, and 75%, respectively. The surprisingly low bootstrap score for ME is related to our complete deletion of gapped positions in the pairwise distance calculations (Fig. 7). Despite this low score, the optimal ME phylogeny still supports as best a Sox9 assignment for the previously undescribed lamprey protein and is therefore consistent with the other phylogenetic results.

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Fig. 6. Extended majority-rule consensus tree for the Bayesian phylogenetics (BP) analysis of the fibril A collagen proteins. Numbers indicate posterior probabilities that are given for each group with >50% credibility and for those clades that are combinable with this first set. Branch lengths are proportional to the means of the posterior probability densities for their expected replacements per site. This tree is rooted by sea urchin ColP2. This root along the Col3 clade illustrates an alternative placement for maximum parsimony (MP), maximum likelihood (ML), and BP relative to the Col5 rooting for minimum evolution (ME) (Fig. 1). In contrast to this position, the latter is consistent with available phylogenetic and linkage data for the physically linked Hox gene clusters (1, 2). Nevertheless, this discrepancy over the root does not diminish the fact that all phylogenetic methods converge onto the same placement of the two lamprey collagens at the base of the Col2a1 clade. Thus, no ambiguity exists over the identification of the two previously undescribed lamprey sequences as orthologues of Col2a1.

Fig. 7. Alignment of inferred amino acid sequence for lamprey Sox9 with chordate SoxE proteins. The inferred amino acid sequence of the previously undescribed lamprey Sox clone most closely matched those of the gnathostome Sox9 family. For example, this lamprey sequence was 96% identical to human Sox9 within the HMG-box domain (highlighted here in yellow). Outside of this domain, the available SoxE proteins often showed considerable length variation, thereby making these regions difficult to align. Such problematic regions were removed before phylogenetic analyses (1), resulting in the multiple sequence alignment shown here with 495 aligned positions. In ME, gapped positions were subjected to complete deletion (i.e., the removal of their entire column in the multiple sequence alignment) to ensure uniform comparisons among all pairwise distances (2). However, this approach also reduced the number of aligned positions for ME to 219 sites, thereby resulting in even less information for the placement of the previously undescribed lamprey sequence at the base of the Sox9 clade (as indicated by its low bootstrap score in Fig. 4). In the end, we based our current assignment of this lamprey sequence on three different lines of evidence (3, 4): (i) on the presence of a Sox9-signature motif (which is located immediately 3' to the HMG-box and is underlined in the figure); (ii) on the consistent phylogenetic placement of the lamprey protein to the base of the Sox9 clade (Fig. 4); and (iii) on its overlapping gene expression pattern with that of Sox9 in jawed vertebrates (see text).

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